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Sep 2017

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A Flow Cytometric Method to Determine Transfection Efficiency
利用流式细胞术测定转染效率   

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Abstract

Mammalian cell transfection is a powerful technique commonly used in molecular biology to express exogenous DNA or RNA in cells and study gene and protein function. Although several transfection strategies have been developed, there is a wide variation with regards to transfection efficiency, cell toxicity and reproducibility. Thus, a sensitive and robust method that can optimize transfection efficiency based not only on expression of the target protein of interest but also on the uptake of the nucleic acids, can be an important tool in molecular biology. Herein, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency while overcoming limitations of prior established methods that quantify transfection efficiency.

Keywords: Transfection (转染), Flow cytometry (流式细胞术), Nucleic acids (核酸), Protein expression (蛋白表达), DNA labeling (DNA标记)

Background

Transfection is one of the most commonly used techniques in molecular biology (Stoll and Calos, 2002; Kim and Eberwine, 2010). Transfection is the process of introducing nucleic acid (DNA that carries a gene of interest or mRNA) into target cells that then eventually express the desired nucleic acid or protein. There are several biological, chemical, and physical methods for introducing nucleic acids into cells (Stoll and Calos, 2002; Kim and Eberwine, 2010; Zhou et al., 2016). However, all these methods are variable and don’t assess the cell transfection efficiency, cell toxicity and the level of gene expression within the same experiment. To truly optimize cellular transfection, a sensitive and robust detection assay is required to quantify and optimize the efficiency of different transfection methods to deliver the target gene into the cytosol and facilitate protein expression, while reducing cell toxicity.

Herein, we demonstrate the development of a flow-cytometric assay to determine transfection efficiency by labeling a reporter plasmid with Label IT® TrackerTM (Homann et al., 2017) (Figure 1). This method does not depend on co-transfection of two different plasmids and simultaneously quantifies cell death, uptake of the labeled plasmid during transient transfection, and expression of the target protein. We demonstrate that this method can be used as a tool to i) optimize transfection efficiency in 2 independent standard cell lines, ii) quantify cellular toxicity of different transfection methods, iii) determine uptake of DNA into difficult to transfect cells via electroporation without the need to use co-transfection of GFP plasmid that can further reduce the efficiency of transfection. This flow cytometric method can be directly applied to optimize several transfection methods including gene therapy strategies (e.g., CRISPR/Cas system).


Figure 1. Experimental design for determination of transfection efficiency by flow cytometric method. The plasmid DNA was labeled with FITC by DNA label IT@ tracker. After transfection, cells were detected by flow cytometry. The FITC fluorochrome is used to detect intracellular levels of the transfected plasmid that has been labeled with FITC (Label IT tracker, green). The second fluorochrome is used to quantify expression of the target protein (by directly measuring fluorescence of the expressed protein if the target protein is fluorescent or by using a fluorescent-labeled antibody against the target protein, red). Either Q1+Q2 (DNA signal) or Q2+Q3 (protein signal) should be used as readouts of transfection efficiency.

Materials and Reagents

  1. 4 mm cuvettes (Gene Pulser cuvettes, Biorad, Hercules, CA)
  2. Cell lines of interest
    1. 293T cell line (ATCC, catalog number: A-498)
    2. Jurkat E6-1 (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIH)
    3. Jurkat Clone E6-1 (from Dr. Arthur Weiss) 
  3. pNL4-3, received from AIDS reagent program (#114, NIH)
  4. pUltraHot encoding mCherry (8,314 bp) was a kind gift of Jeff F. Miller, California Nano Systems Institute
  5. One ShotTM Stbl3TM Chemically Competent E. coli (Thermo Scientific, catalog number: C737303)
  6. Stellar bacteria (Clontech, catalog number: 636763) 
  7. Dulbecco's modified Eagle medium (DMEM) (Thermo Scientific, catalog number: 11965-092) 
  8. Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Scientific, catalog number: 61870127)
  9. Fetal bovine serum (FBS) (Omega Scientific, catalog number: FB-02)
  10. OptiMEM (Thermo Scientific, catalog number: 31985-070)
  11. Penicillin and streptomycin (Thermo Scientific, catalog number: 15-140-122) 
  12. L-glutamine (Invitrogen, catalog number: 125030-081)
  13. Phosphate-buffered saline (PBS) (Omega Scientific, catalog number: FB-02)
  14. 4% paraformaldehyde (PFA) (Thermo Scientific, catalog number: J19943-K2)
  15. Tween 20 (Sigma, catalog number: P9416)
  16. PureLink HiPure Midiprep Kit (Thermo Scientific, catalog number: K210004)
  17. DNA Label IT® TrackerTM [Fluorescein isothiocyanate (FITC)] (Mirus, catalog number: MIR7025)
  18. TransIT-X2 (Mirus, catalog number: MIR6004)
  19. Jet Prime (Polyplus, catalog number: 114-07)
  20. Lipofectamine 2000 (Thermo Scientific, catalog number: 11668019)
  21. Fugene HD (Promega, catalog number: E2311)
  22. Quantum Molecules of Equivalent Soluble Fluorochrome (MESF) (Bangs Laboratories, catalog number: 647-B)
  23. Ghost Violet 450 Live/dead dye (Tonbo Biosciences, catalog number: 13-0863-T100)
  24. Anti-human immunodeficiency virus (HIV)-1 p24 monoclonal antibody (71-31) (NIH AIDS reagent program #530)
  25. Human IgG1 (Biolegend, catalog number: 409302)
  26. Mix-N-Stain CF647 dye (Biotium, catalog number: 92238)
  27. 10% FBS complete culturing medium for 293T (see Recipes)
  28. 10% FBS complete culturing medium for Jurkat cells (see Recipes)
  29. 0.02% Tween/PBS (see Recipes)

Equipment

  1. 37 °C, 5% CO2 humidified incubator (Thermoforma, model: 3110)
  2. Centrifuge (Thermo scientific, model: ST8)
  3. BioRad Gene Pulser Xcell electroporation system
  4. LSRII Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA)
  5. Spectrophotometer (Thermo Fisher, NanoDropTM 2000)

Software

  1. Acquisition Software: BDFACSDiVaTM Software (BD Biosciences)
  2. Analysis Software: FlowJo version 10.6

Procedure

  1. Plasmid labeling
    1. Extract pNL4-3 and pUltraHot encoding mCherry for transfection from Stbl3TM (Thermo Fisher, Waltham, MA) or Stellar (Clontech, Mountain View, CA) bacteria using the PureLink HiPure Midiprep Kit (Thermo Fisher, Waltham, MA). Determine concentration and purity (260/280 ratio) of nucleic acid using spectrophotometry. 
    2. Label Plasmid DNA with DNA Label IT® Tracker (Mirus, Madison, WI) the day before transfection using 0.5 μl FITC/1 μg DNA according to the manufacturer’s protocol. Remove unreacted Label IT® Tracker reagent using ethanol precipitation. According to the manufacturer, 1 μl of labeling dye per 1 μg plasmid DNA will yield labeling efficiencies of approximately one Label molecule every 40 base pairs (on average) of double-stranded DNA. Determine the purity and concentration of labeled DNA using spectrophotometry.

  2. Antibody conjugation
    1. Conjugate Anti-HIV-1 p24 monoclonal antibody (71-31) with Mix-n-Stain CF647 dye, according to the manufacturer’s protocol. 
    2. Conjugate Human IgG1 control with Mix-n-Stain CF647 dye according to the manufacturer’s protocol.

  3. Transfection of 293T cells with FITC labeled DNA by different transfection reagents
    1. Plate 3 x 105 293T cells of < 20 passages into 12-well plates the day before transfection. At this density, cells are usually attached to the bottom of the well at a confluency of 70%-80% after 24 h.
    2. Twenty-four hours after seeding, transfect cells with FITC-labeled and unlabeled pNL4-3 or pUltraHot encoding mCherry. The transfections can be performed in 12-well plates using 1 μg plasmid DNA and 3 μl TransIT X2 in serum-free media, according to the manufacturer’s protocol.
    3. Culture cells in complete media after adding transfection complexes for 6 h. Then change medium to 10% FBS complete culturing medium.
      Note: For experiments comparing different transfection reagents, the cells can be transfected with 1 μg DNA per well with varying amounts of transfection reagents to facilitate the recommended ratio of transfection reagent to DNA for each reagent: 3 μl TransIT X2, 4 μl Lipofectamine 2000, 2 μl Jet Prime and 3 μl Fugene HD. 
    4. Harvest cells at 24 h post-transfection and wash one time in PBS.
      Note: For time course experiments, the cells can be transfected at the same time and harvested at 0, 6, 12, 24, 36, 48 and 72 h.
    5. Fix cells in 4% PFA for 15 min at room temperature.
    6. Permeabilize cells in 0.2% Tween/PBS for 15 min. 
    7. Stain cells for HIV-1 p24 protein with 1 μg anti-p24 antibody conjugated to CF647 in 100 μl staining medium containing PBS/2% BSA for 30 min at 4 °C.
    8. Wash 293T cells once with PBS.
    9. Fix cells in 4% PFA for 15 min.

  4. Electroporation of Jurkat cells with FITC labeled pUltraHot
    1. Wash Jurkat E6-1 cells 2 times in OptiMEM before resuspending cells in OptiMEM at 1 x 106 cells/100 μl. 
    2. Transfer 1 x 106 cells into 4 mm cuvettes and add 4 μg of labeled or unlabeled pUltraHot plasmid to the suspension.
    3. Electroporate Jurkat E6-1 cells with a BioRad Gene Pulser Xcell electroporation system using exponential protocol with 250 voltage, 350 μF capacitance, 1,000 Resistance at room temperature.
    4. Transfer cells immediately into pre-warmed media and incubate cells for 24 h before staining for viability and Flow cytometric analysis. 
    5. Collect Jurkat cells transfected with pUltraHot. Stain Jurkat cells with 1 μl Ghost Violet 450 Live/dead dye in 1 ml PBS incubate 30 min at room temperature. 
    6. Wash Jurkat cells with 1% FBS in PBS. 
    7. Fix Jurkat cells in 4% PFA for 15 min.

  5. Flow cytometry analysis
    1. Acquire samples on an LSRII Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) with BDFACSDiVaTM Software (BD Biosciences). The flow cytometer is equipped with 405, 488, 561 and 635 nm lasers, and emission filters for Pacific blue (LP: −, BP:450/50), Alexa fluor-488 (LP: 505, BP: 530/30), PE (LP: −, BP: 582/15), mCherry (LP: 600 BP: 610/20), PerCP-Cy5.5 (LP: 685, BP: 695/40), APC (LP: −, BP: 670/14). FITC fluorescence intensity (y-axis) is plotted on a log scale against the fluorescence intensity (x-axis) of fluorochrome that is used to quantify protein expression (e.g., mCherry) (Figure 2).
    2. The mean fluorescence intensity (MFIs) of FITC and Alexa Fluor® 647 can be further standardized using Quantum MESF (Molecules of Equivalent Soluble Fluorochrome) kits for Alexa Fluor® 488 and Alexa Fluor® 647 as previously described (Kapoor et al., 2009).


      Figure 2. Flow cytometric determination of transfection efficiency based on two independent readouts (DNA plasmid uptake and protein expression). Representative transfections are shown. 293T cells underwent chemical transfection using the TransITX2 transfection reagent as described in Procedure. The same amount (1 μg) of DNA was used for two independent plasmids: a small (B. pUltraHot expressing mCherry, 8.3 kb) and a large (C. pNL4-3 expressing p24, 14.0 kb) DNA plasmid. Gating strategy is shown: A) forward and side scatters B) discrimination of doublets C, D) two independent readouts of transfection efficiency. FITC fluorescence corresponds to the uptake of FITC-labeled plasmid DNA (y-axis). A fluorochrome that has no spectral overlap with FITC is used to quantify protein expression. Either a fluorescent protein can be used (e.g., mCherry; shown in C) or a protein labeled with a fluorescent-labeled antibody (e.g., intracellular expression of HIV-1 p24 protein was detected by a CF647-labeled anti-p24 antibody; shown in D). Co-expression of DNA taken up by cells and target protein were analyzed 24 h after transfection. The numbers in the quadrants indicate the percentages of viable cells that took up the FITC labeled DNA plasmid versus the expressed protein that was detected. The following dot plots are shown for each chemical transfection in 293T cells: i) untransfected cells (negative control), ii) cells transfected with FITC-labeled DNA plasmid harvested before protein expression occurred (3 h post transfection), iii) cells transfected with unlabeled plasmid harvested 24 h after transfection (when protein expression can be quantified) iv) cells transfected with FITC-labeled DNA plasmid and harvested 24 h after transfection (when protein expression can be quantified). In this plot Q3 quadrant demonstrates many cells that express protein but do not show any fluorescence associated with uptake of the plasmid DNA. Either Q1+Q2 (DNA signal) or Q2+Q3 (protein signal) should be used as readouts of transfection efficiency. E. Transfection efficiency was quantified in human lymphocytes (Jurkat E6 cells) harvested 24 h after electroporation with FITC-labeled DNA mCherry plasmid without the need to use co-transfection of 2 different plasmids and GFP reporter.

Data analysis

Results of the peak fluorescence measurements are blank-corrected with signal from non-transfected cells and normalized to 1 x 104 cells with the corresponding cell density value from the same well. Values are presented as max fluorescence (median fluorescence intensity; MFI) or relative fluorescence units (rfu) per 10 cells. Cell viability at 24 and 48 h is expressed as % of viable cells (based on the death dye) of total number of cells and can be compared to the viability from the non-transfected control cells. This method takes into consideration cell toxicity as a direct result of the transfection and the nucleic acid per se and uses two independent readouts of transfection efficiency: a) the amount of plasmid nucleic acid that cells have taken up during transfection b) the amount of the encoded expressed protein. In summary, we provide a relatively simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency.

Recipes

  1. 10% FBS complete culturing medium for 293T
    DMEM supplemented with:
    10% heat-inactivated FBS
    100 unit/ml Penicillin and streptomycin
    2 mM L-glutamine
  2. 10% FBS complete culturing medium for Jurkat cells
    RPMI1640 supplemented with:
    10% heat-inactivated FBS
    100 unit/ml Penicillin and Streptomycin
    2 mM L-glutamine
  3. 0.02% Tween/PBS
    4 μl of Tween 20 in 20 ml PBS

Acknowledgments

We would like to acknowledge the Center for AIDS Research Virology Core Lab and the Gene and Cellular Therapy Core that are supported by the National Institutes of Health [UCLA Center for AIDS Research (CFAR) NIH/NIAID 5P30 AI028697] and by the UCLA AIDS Institute and the UCLA Council of Bioscience Resources. This research was supported by NIH grants NIH/NCATS grant # UL1TR000124, NIH K08AI08272 and by grant from the UCLA AIDS Institute/UCLA Center for AIDS Research (CFAR) (NIH/NIAID AI028697). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or any of the funders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests

The authors have declared that no competing interests exist.

References

  1. Stoll, S. M. and Calos, M. P. (2002). Extrachromosomal plasmid vectors for gene therapy. Curr Opin Mol Ther 4(4): 299-305.
  2. Kim, T. K. and Eberwine, J. H. (2010). Mammalian cell transfection: the present and the future. Anal Bioanal Chem 397(8): 3173-3178.
  3. Zhou, Z. L., Sun, X. X., Ma, J., Man, C. H., Wong, A. S., Leung, A. Y. and Ngan, A. H. (2016). Mechanical oscillations enhance gene delivery into suspended cells. Sci Rep 6: 22824.
  4. Homann, S., Hofmann, C., Gorin, A. M., Nguyen, H. C. X., Huynh, D., Hamid, P., Maithel, N., Yacoubian, V., Mu, W., Kossyvakis, A., Sen Roy, S., Yang, O. O. and Kelesidis, T. (2017). A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells. PLoS One 12(9): e0182941.
  5. Kapoor, V., Hakim, F. T., Rehman, N., Gress, R. E. and Telford, W. G. (2009). Quantum dots thermal stability improves simultaneous phenotype-specific telomere length measurement by FISH-flow cytometry. J Immunol Methods 344(1): 6-14.

简介

摘要:哺乳动物细胞转染是分子生物学中常用的一种强有力的技术,用于在细胞中表达外源DNA或RNA并研究基因和蛋白质功能。 尽管已经开发了几种转染策略,但在转染效率,细胞毒性和再现性方面存在很大差异。 因此,不仅可以基于目标靶蛋白的表达而且还可以基于核酸的摄取来优化转染效率的灵敏且稳健的方法可以是分子生物学中的重要工具。 在这里,我们提出了一种简单,快速和稳健的流式细胞术方法,可用作优化转染效率的工具,同时克服先前确定的量化转染效率的方法的局限性。


背景:转染是分子生物学中最常用的技术之一(Stoll和Calos,2002; Kim和Eberwine,2010)。转染是将核酸(携带目的基因或mRNA的DNA)引入靶细胞然后最终表达所需核酸或蛋白质的过程。将核酸引入细胞有几种生物,化学和物理方法(Stoll和Calos,2002; Kim和Eberwine,2010; Zhou et al。,2016)。然而,所有这些方法都是可变的,并且不评估同一实验中的细胞转染效率,细胞毒性和基因表达水平。为了真正优化细胞转染,需要灵敏且稳健的检测分析来量化和优化不同转染方法的效率,以将靶基因递送到胞质溶胶中并促进蛋白质表达,同时降低细胞毒性。

在这里,我们通过标记IT ® Tracker TM 标记报告质粒来证明转染效率的流式细胞分析的开发(Homann et al。,2017)(图1)。该方法不依赖于两种不同质粒的共转染,同时定量细胞死亡,瞬时转染期间标记质粒的摄取和靶蛋白的表达。我们证明该方法可用作以下工具:i)优化2种独立标准细胞系的转染效率; ii)量化不同转染方法的细胞毒性; iii)通过电穿孔确定难以转染细胞的DNA摄取而无需使用可以进一步降低转染效率的GFP质粒的共转染。该流式细胞术方法可以直接应用于优化几种转染方法,包括基因治疗策略(例如,CRISPR / Cas系统)。


图1.通过流式细胞术确定转染效率的实验设计通过DNA标记IT @ tracker用FITC标记质粒DNA。转染后,通过流式细胞术检测细胞。 FITC荧光染料用于检测已用FITC标记的转染质粒的细胞内水平(标签IT跟踪器,绿色)。第二荧光染料用于定量靶蛋白的表达(如果靶蛋白是荧光的,则通过直接测量表达的蛋白的荧光或通过使用针对靶蛋白的荧光标记的抗体,红色)。应使用Q1 + Q2(DNA信号)或Q2 + Q3(蛋白质信号)作为转染效率的读数。

关键字:转染, 流式细胞术, 核酸, 蛋白表达, DNA标记

材料和试剂

  1. 4毫米比色皿(Gene Pulser比色皿,Biorad,Hercules,CA)
  2. 感兴趣的细胞系
    1. 293T细胞系(ATCC,目录号:A-498)
    2. Jurkat E6-1(通过NIH艾滋病试剂计划,NIH艾滋病科获得)
    3. Jurkat Clone E6-1(来自Arthur Weiss博士)&nbsp;
  3. pNL4-3,来自艾滋病试剂计划(#114,NIH)
  4. pUltraHot编码mCherry(8,314 bp)是加州纳米系统研究所Jeff F. Miller的礼物
  5. One Shot TM Stbl3 TM 化学能力 E.大肠杆菌(Thermo Scientific,目录号:C737303)
  6. 恒星细菌(Clontech,目录号:636763)&nbsp;
  7. Dulbecco的改良Eagle培养基(DMEM)(Thermo Scientific,目录号:11965-092)&nbsp;
  8. Roswell Park Memorial Institute(RPMI)1640培养基(Thermo Scientific,目录号:61870127)
  9. 胎牛血清(FBS)(Omega Scientific,目录号:FB-02)
  10. OptiMEM(Thermo Scientific,目录号:31985-070)
  11. 青霉素和链霉素(Thermo Scientific,目录号:15-140-122)&nbsp;
  12. L-谷氨酰胺(Invitrogen,目录号:125030-081)
  13. 磷酸盐缓冲盐水(PBS)(Omega Scientific,目录号:FB-02)
  14. 4%多聚甲醛(PFA)(Thermo Scientific,目录号:J19943-K2)
  15. 吐温20(西格玛,目录号:P9416)
  16. PureLink HiPure Midiprep Kit(Thermo Scientific,目录号:K210004)
  17. DNA标签IT ® Tracker TM [异硫氰酸荧光素(FITC)](Mirus,目录号:MIR7025)
  18. TransIT-X2(Mirus,目录号:MIR6004)
  19. Jet Prime(Polyplus,目录号:114-07)
  20. Lipofectamine 2000(Thermo Scientific,目录号:11668019)
  21. Fugene HD(Promega,目录号:E2311)
  22. 等效可溶性荧光素(MESF)的量子分子(Bangs Laboratories,目录号:647-B)
  23. Ghost Violet 450活/死染料(Tonbo Biosciences,目录号:13-0863-T100)
  24. 抗人类免疫缺陷病毒(HIV)-1 p24单克隆抗体(71-31)(NIH AIDS试剂计划#530)
  25. 人IgG1(Biolegend,目录号:409302)
  26. Mix-N-Stain CF647染料(Biotium,目录号:92238)
  27. 用于293T的10%FBS完全培养基(参见食谱)
  28. 用于Jurkat细胞的10%FBS完全培养基(参见食谱)
  29. 0.02%吐温/ PBS(见食谱)

设备

  1. 37°C,5%CO 2 加湿培养箱(Thermoforma,型号:3110)
  2. 离心机(Thermo scientific,型号:ST8)
  3. BioRad Gene Pulser Xcell电穿孔系统
  4. LSRII Fortessa流式细胞仪(BD Biosciences,San Jose,CA,USA)
  5. 分光光度计(Thermo Fisher,NanoDrop TM 2000)

软件

  1. 采集软件:BDFACSDiVa TM 软件(BD Biosciences)
  2. 分析软件:FlowJo版本10.6

程序

  1. 质粒标记
    1. 使用PureLink HiPure Midiprep试剂盒(Thermo Fisher,Waltham)从stbl3 TM (Thermo Fisher,Waltham,MA)或Stellar(Clontech,Mountain View,CA)细菌中提取编码mCherry的pNL4-3和pUltraHot进行转染, 嘛)。使用分光光度法测定核酸的浓度和纯度(260/280比率)。&nbsp;
    2. 根据制造商的方案,使用0.5μlFITC/1μgDNA在转染前一天用DNA标签IT ® Tracker(Mirus,Madison,WI)标记质粒DNA。使用乙醇沉淀去除未反应的标签IT ®跟踪试剂。根据制造商,每1μg质粒DNA1μl标记染料将产生每40碱基对(平均)双链DNA约一个Label分子的标记效率。用分光光度法测定标记DNA的纯度和浓度。

  2. 抗体结合
    1. 根据制造商的方案,使用Mix-n-Stain CF647染料缀合抗HIV-1 p24单克隆抗体(71-31)。&nbsp;
    2. 根据制造商的方案,用Mix-n-Stain CF647染料缀合人IgG1对照。

  3. 用不同的转染试剂用FITC标记的DNA转染293T细胞
    1. 板3×10 5 293T细胞<1。转染前一天将20次传代到12孔板中。在该密度下,细胞通常在24小时后以70%-80%的汇合度附着于孔的底部。
    2. 接种后二十四小时,用FITC标记的和未标记的pNL4-3或编码mCherry的pUltraHot转染细胞。根据制造商的方案,转染可以在12孔板中使用1μg质粒DNA和3μlTransITX2在无血清培养基中进行。
    3. 加入转染复合物6小时后,在完全培养基中培养细胞。然后将培养基更换为10%FBS完全培养基。
      注意:对于比较不同转染试剂的实验,可以用每孔1μgDNA转染不同量的转染试剂,以促进每种试剂的转染试剂与DNA的推荐比例:3μlTransITX2,4μl Lipofectamine2000,2μlJetPrime和3μlFugeneHD。&nbsp;
    4. 转染后24小时收获细胞并在PBS中洗涤一次。
      注意:对于时间过程实验,细胞可以同时转染,并在0,6,12,24,36,48和72小时收获。
    5. 在室温下将细胞在4%PFA中固定15分钟。
    6. 将细胞在0.2%吐温/ PBS中渗透15分钟。&nbsp;
    7. 用含有PBS / 2%BSA的100μl染色培养基中的1μg抗-p24抗体与CF647缀合,在4℃下染色HIV-1 p24蛋白的细胞30分钟。
    8. 用PBS洗涤293T细胞一次。
    9. 将细胞在4%PFA中固定15分钟。

  4. 用FITC标记的pUltraHot电穿孔Jurkat细胞
    1. 在OptiMEM中洗涤Jurkat E6-1细胞2次,然后在OptiMEM中以1×10 6 细胞/100μl重悬细胞。&nbsp;
    2. 将1×10 6个细胞转移到4mm比色杯中,并向悬浮液中加入4μg标记或未标记的pUltraHot质粒。
    3. 使用BioRad Gene Pulser Xcell电穿孔系统电穿孔Jurkat E6-1细胞,使用指数方案,250电压,350μF电容,室温下1,000电阻。
    4. 将细胞立即转移到预热的培养基中并孵育细胞24小时,然后染色以获得活力和流式细胞术分析。&nbsp;
    5. 收集用pUltraHot转染的Jurkat细胞。用1μlGhostViolet 450活菌/死染料在1 ml PBS中染色Jurkat细胞,在室温下孵育30分钟。&nbsp;
    6. 用PBS中的1%FBS洗涤Jurkat细胞。&nbsp;
    7. 将Jurkat细胞在4%PFA中固定15分钟。

  5. 流式细胞术分析
    1. 用BDFACSDiVa TM 软件(BD Biosciences)在LSRII Fortessa流式细胞仪(BD Biosciences,San Jose,CA,USA)上获取样品。流式细胞仪配备405,488,561和635 nm激光器,太平洋蓝光发射滤光片(LP: - ,BP:450/50),Alexa fluor-488(LP:505,BP:530/30), PE(LP: - ,BP:582/15),mCherry(LP:600 BP:610/20),PerCP-Cy5.5(LP:685,BP:695/40),APC(LP: - ,BP:十四分之六百七十〇)。 FITC荧光强度( y -axis)以对数标度绘制,相对于荧光染料的荧光强度(x轴),用于定量蛋白质表达(例如,mCherry )(图2)。
    2. FITC和Alexa Fluor ® 647的平均荧光强度(MFIs)可以使用Quantum MESF(等效可溶性荧光染料分子)试剂盒进一步标准化,用于Alexa Fluor ® 488和Alexa如前所述的Fluor ® 647(Kapoor et al。,2009)。


      图2.基于两个独立的读数(DNA质粒摄取和蛋白质表达)的流式细胞术确定转染效率。显示了代表性的转染。如过程中所述,使用TransITX2转染试剂对293T细胞进行化学转染。将相同量(1μg)的DNA用于两种独立的质粒:小的(表达mCherry的B.pUltraHot,8.3kb)和大的(表达p.LN4-4的p24,14.0kb)DNA质粒。显示门控策略:A)前向和侧向散射B)双重C,D的鉴别两个独立的转染效率读数。 FITC荧光对应于FITC标记的质粒DNA的摄取(y轴)。与FITC没有光谱重叠的荧光染料用于定量蛋白质表达。可以使用荧光蛋白(例如,mCherry;显示在C中)或用荧光标记的抗体标记的蛋白质(例如,HIV-1 p24的细胞内表达)通过CF647标记的抗p24抗体检测蛋白质;显示在D)中。转染后24小时分析细胞和靶蛋白吸收的DNA的共表达。象限中的数字表示摄取FITC标记的DNA质粒的活细胞与检测到的表达蛋白的百分比。对于293T细胞中的每次化学转染显示以下点图:i)未转染的细胞(阴性对照),ii)在蛋白质表达发生之前(转染后3小时)收获的用FITC标记的DNA质粒转染的细胞,iii)用转染的细胞转染的细胞转染后24小时收获未标记的质粒(当可以定量蛋白质表达时)iv)用FITC标记的DNA质粒转染的细胞,并在转染后24小时收获(当可以定量蛋白质表达时)。在该图中,Q3象限显示许多表达蛋白质但未显示与质粒DNA摄取相关的荧光的细胞。应使用Q1 + Q2(DNA信号)或Q2 + Q3(蛋白质信号)作为转染效率的读数。 E.在用FITC标记的DNA mCherry质粒进行电穿孔后24小时收获的人淋巴细胞(Jurkat E6细胞)中定量转染效率,无需使用2种不同质粒和GFP报告基因的共转染。

数据分析

用来自未转染细胞的信号对峰值荧光测量结果进行空白校正,并将其标准化为1×10 6个 4 细胞,具有来自相同孔的相应细胞密度值。数值表示为每10个细胞的最大荧光(中值荧光强度; MFI)或相对荧光单位(rfu)。 24和48小时的细胞活力表示为细胞总数的活细胞%(基于死亡染料),并且可以与来自未转染的对照细胞的活力进行比较。该方法考虑到细胞毒性作为转染的直接结果和核酸本身并且使用两个独立的转染效率读数:a)细胞在细胞摄取过程中吸收的质粒核酸的量转染b)编码的表达蛋白的量。总之,我们提供了一种相对简单,快速和稳健的流式细胞术方法,可用作优化转染效率的工具。

食谱

  1. 10%FBS完全培养基293T
    DMEM补充:
    10%热灭活FBS
    100单位/ ml青霉素和链霉素
    2mM L-谷氨酰胺
  2. 用于Jurkat细胞的10%FBS完全培养基
    RPMI1640补充:
    10%热灭活FBS
    100单位/ ml青霉素和链霉素
    2mM L-谷氨酰胺
  3. 0.02%吐温/ PBS
    在20ml PBS中的4μl吐温20

致谢

我们要感谢由美国国立卫生研究院[加州大学洛杉矶分校艾滋病研究中心(CFAR)NIH / NIAID 5P30 AI028697]和加州大学洛杉矶分校艾滋病研究所支持的艾滋病研究病毒学核心实验室和基因和细胞治疗核心中心。和加州大学洛杉矶分校生物科学资源委员会。 NIH授予NIH / NCATS拨款#UL1TR000124,NIH K08AI08272以及加州大学洛杉矶分校艾滋病研究所/加州大学洛杉矶分校艾滋病研究中心(CFAR)(NIH / NIAID AI028697)的资助,支持该研究。内容完全由作者负责,并不一定代表美国国立卫生研究院或任何资助者的官方观点。资助者在研究设计,数据收集和分析,决定发表或准备手稿方面没有任何作用。

利益争夺

作者宣称没有竞争利益存在。

参考

  1. Stoll,S。M.和Calos,M。P.(2002)。 用于基因治疗的染色体外质粒载体。 Curr Opin Mol Ther 4(4):299-305。
  2. Kim,T.K。和Eberwine,J。H.(2010)。 哺乳动物细胞转染:现在和将来。 Anal Bioanal Chem 397(8):3173-3178。
  3. Zhou,Z.L.,Sun,X.X.,Ma,J.,Man,C.H。,Wong,A.S.,Leung,A.Y。和Ngan,A.H。(2016)。 机械振荡可增强基因向悬浮细胞的传递。 Sci Rep 6:22824。
  4. Homann,S.,Hofmann,C.,Gorin,AM,Nguyen,HCX,Huynh,D.,Hamid,P.,Maithel,N.,Yacoubian,V.,Mu,W.,Kossyvakis,A.,Sen Roy ,S.,Yang,OO和Kelesidis,T。(2017)。 一种新型快速,可重复的流式细胞仪方法,用于优化细胞中的转染效率。 PLoS One 12(9):e0182941。
  5. Kapoor,V.,Hakim,F.T.,Rehman,N.,Gress,R。E.和Telford,W。G.(2009)。 量子点热稳定性通过FISH流式细胞仪改善了同时表型特异性的端粒长度测量。 J Immunol Methods 344(1):6-14。
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引用:Mu, W., Homann, S., Hofmann, C., Gorin, A., Huynh, D., Yang, O. O. and Kelesidis, T. (2019). A Flow Cytometric Method to Determine Transfection Efficiency. Bio-protocol 9(10): e3244. DOI: 10.21769/BioProtoc.3244.
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